The polymerase chain reaction (PCR) is widely used to amplify stretches of DNA, including cDNA reverse transcribed from RNA, for assays for diagnostic and other purposes. See U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,965,188. See, generally, PCR PROTOCOLS, a Guide to Methods and Applications, Innis et al. eds., Academic Press (San Diego, Calif. (USA) 1990). PCR reactions are generally designed to be symmetric, that is, to make double-stranded copies by utilizing a forward primer and a reverse primer in equimolar concentrations. The two primers are designed to have “melting temperatures,” or “Tm's” that are “balanced” (Innis et al., page 9), which is generally understood to mean equal or within a few degrees (° C.) of one another. A commonly used computer software program for primer design warns users to avoid high Tm difference, and has an automatic Tm matching feature. (Oligo® Primer Analysis Software Manual, version 6.0 for Windows, Molecular Biology Insights, Inc., Sixth Edition, March 2000). The Tm's of linear primers comprised of deoxyribonucleotides (DNA) have been commonly determined by the “percent GC” method (Innis et al., page 9) or the “2 (A+T) plus 4 (G+C)” method (Wallace et al. (1979) “Hybridization of Synthetic Oligodeoxyribonucletides to phi chi 174 DNA: the Effect of a Single Base Pair Mismatch,” Nucleic Acids Res. 6 (11): 35433557) or the “Nearest Neighbor” method (SantaLucia, J. (1998) “A Unified view of Paymer, Dumbbell, and Oligonucleotide DNA Nearest Neighbor Thermodynamics,” Proc. Natl. Acad. Sci. USA 95: 1460-1465; Allawi, H. T. and SantaLucia, J. (1997) “Thermodynamics and NMR of Internal G·T Mismatches In DNA,” Biochem. 36: 10581-10594).
PCR is a repeated series of steps of denaturation, or strand melting, to create single-stranded templates; primer annealing; and primer extension by a thermally stable DNA polymerase such as Thermus aguaticus (Taq) DNA polymerase. A typical three-step PCR protocol (see Innis et al., Chapter 1) may include denaturation, or strand melting, at 93-95° C. for more than 5 sec, primer annealing at 55-65° C. for 10-60 sec, and primer extension for 15-120 sec at a temperature at which the polymerase is highly active, for example, 72° C. for Taq DNA polymerase. A typical two-step PCR protocol may differ by having the same temperature for primer annealing as for primer extension, for example, 60° C. or 72° C. For either three-step PCR or two-step PCR, an amplification involves cycling the reaction mixture through the foregoing series of steps numerous times, typically 25-40 times. During the course of the reaction the times and temperatures of individual steps in the reaction may remain unchanged from cycle to cycle, or they may be changed at one or more points in the course of the reaction to promote efficiency or enhance selectivity. In addition to the pair of primers and target nucleic acid a PCR reaction mixture typically contains each of the four deoxyribonucleotide 5′ triphosphates (dNTPs) at equimolar concentrations, a thermostable polymerase, a divalent cation, and a buffering agent. A reverse transcriptase is included for RNA targets, unless the polymerase possesses that activity. The volume of such reactions is typically 25-100 μl. Multiple target sequences can be amplified in the same reaction. In the case of cDNA amplification, PCR is preceded by a separate reaction for reverse transcription of RNA into cDNA, unless the polymerase used in the PCR possesses reverse transcriptase activity. The number of cycles for a particular PCR amplification depends on several factors including: a) the amount of the starting material, b) the efficiency of the reaction, and c) the method and sensitivity of detection or subsequent analysis of the product. Cycling conditions, reagent concentrations, primer design, and appropriate apparatuses for typical cyclic amplification reactions are well known in the art (see, for example, Ausubel, F. Current Protocols in Molecular Biology (1988) Chapter 15: “The Polymerase Chain Reaction,” J. Wiley (New York, N.Y. (USA)).
Ideally, each strand of each amplicon molecule binds a primer at one end and serves as a template for a subsequent round of synthesis. The rate of generation of primer extension products, or amplicons, is thus exponential, doubling during each cycle. The amplicons include both plus (+) and minus (−) strands, which hybridize to one another to form double strands. To differentiate typical PCR from special variations described herein, we refer to typical PCR as “symmetric” PCR. Symmetric PCR thus results in an exponential increase of one or more double-stranded amplicon molecules, and both strands of each amplicon accumulate in equal amounts during each round of replication. The efficiency of exponential amplification via symmetric PCR eventually declines, and the rate of amplicon accumulation slows down and stops. Kinetic analysis of symmetric PCR reveals that reactions are composed of: a) an undetected amplification phase (initial cycles) during which both strands of the target sequence increase exponentially, but the amount of the product thus far accumulated is below the detectable level for the particular method of detection in use; b) a detected amplification phase (additional cycles) during which both strands of the target sequence continue to increase in parallel and the amount of the product is detectable; c) a plateau phase (terminal cycles) during which synthesis of both strands of the amplicon gradually stops and the amount of product no longer increases. Symmetric reactions slow down and stop because the increasing concentrations of complementary amplicon strands hybridize to each other (reanneal), and this out-competes the ability of the separate primers to hybridize to their respective target strands. Typically reactions are run long enough to guarantee accumulation of a detectable amount of product, without regard to the exact number of cycles needed to accomplish that purpose.
Analysis of the amplified product is done by any of several means. For instance, gel electrophoresis or, more recently, capillary electrophoresis has been widely used to separate amplified target sequences, or “amplicons”, according to size. Bands on a gel are typically made visible by use of an intercalating dye, such as ethidium bromide or SYBR® Green, or by transferring the nucleic acid to a membrane and then visualizing it with a radioactively or fluorescently labeled hybridization probe. Analysis by sequencing most commonly involves further amplification, using one primer in each of four reaction vessels together with a different dideoxy dNTP. Under these conditions each reaction generates a linear amplification product comprised of a set of oligonucleotides ending in A, T, C or G depending on which dideoxy dNTP was included in the reaction. See, for example, U.S. Pat. No. 5,075,216.
“Real-time” PCR refers to PCR reactions in which a reporter, typically a fluorescent moiety, is present to monitor the accumulation of the amplicon by a change in signal during the reaction. Such moieties include an intercalating dye, such as SYBR® Green, or a hybridization probe (whether or not extendable as a primer). One real-time PCR method, the 5′ nuclease process, utilizes labeled linear probes, for example dual fluorescent labeled probes (“TaqMan™ probes”), that are digested by the DNA polymerase during the primer extension step, resulting in a detectable signal change (see U.S. Pat. Nos. 5,210,015, 5,487,972 and 5,538,848). Another method utilizes a dye that fluoresces when in contact with double-stranded DNA (see U.S. Pat. No. 5,994,056). A third method utilizes dual fluorescent labeled probes such as “molecular beacon probes”, which are hairpin probes having a fluorophore at one end and a quencher at the other end, and which open and fluoresce when hybridized to their target sequence (see U.S. Pat. Nos. 5,925,517, 6,103,476 and 6,365,729). Other fluorescent labeled probes useful for real-time PCR include Scorpion primers, (primers that have a hairpin probe sequence (containing a fluorophore and a quencher moieties located in close proximity on the hairpin stem) linked to their 5′ end via a PCR stopper such that fluorescence occurs only when the specific probe sequence binds to its complement within the same strand of DNA after extension of the primers during PCR; Whitcombe et al. (1999) “Detection of PCR Products Using Self-Probing Amplicons and Fluorescence,” Nat. Biotechnol. 17: 804-807), Amplifluor primers (primers that have a hairpin probe sequence (containing a fluorophore and a quencher moieties located in close proximity on the hairpin stem) linked to their 5′ end such that fluorescence occurs only when the hairpin unfolds upon replication of the primer following its incorporation into an amplification product; Nazarenko et al. (1997) “A Closed Tube Format for Amplification and Detection of DNA Based on Energy Transfer,” Nucleic Acids Res. 15: 2516-21, Eclipse probes (linear DNA probes that have a minor-groove binding (MGB) protein-quencher complex positioned at the 5′-end of the probe and a fluorophore located at the 3′-end of the probe such that fluorescence only occurs when the probe anneals to a target sequence aided by the MGB protein binding to NDA and the quencher moves away from the fluorophore, (Afonina et al., (2002) “Minor Groove Binder-Conjugated DNA Probes for Quantitative DNA Detection by Hybridization-Triggered Fluorescence,” Biotechniques 32: 946-9), FRET probes (a pair of random coil, or linear, probes, each of which is fluorescently labeled, that hybridize adjacently on a target sequence, causing their labels to interact by fluorescence resonance energy transfer (“FRET”) and produce a detectable signal change), and double-stranded fluorescent probes, (Li, Q. et al. (2002) “A New Class of Homogeneous Nucleic Acid Probes Based on Specific Displacement Hybridization,” Nucl. Acid Res. 30: (2)e5). Probes that are not to be cut, hydrolyzed, or extended (that is, probes that are not primers) are typically designed to disengage from their template either prior to or during the primer extension step of PCR so they will not interfere with this phase of the reaction. For probes such as molecular beacon probes, the melting temperature of the probe is generally 7-10° C. higher than the temperature used to anneal the primers. In practice this means that the melting temperature of the probe is higher than the melting temperature of the primer which hybridizes to the same strand as the probe (Mackay, I. M. (2002) “Survey and Summary: Real-time PCR in Virology”, Nucleic Acids Res. 30(6): 1292-1305). Thus, as the temperature of the reaction is cooled following strand-melting at 95° C. the probe hybridizes to its target strand (hereafter (+) strand) followed by hybridization of the primer for the (+) strand, as the reaction approaches the annealing temperature. As the reaction is warmed again at the end of the annealing step the probe should fall off of the (+) strand while the primer extends along the (+) strand. Thus, the intent is that the probe should not interfere with primer extension. Hybridization and extension of the other primer on the complementary (−) strand also takes place during these steps. A second probe targeted to the (−) strand may also be present in the reaction.
A technique that has found limited use for making single-stranded DNA directly in a PCR reaction is “asymmetric PCR.” Gyllensten and Erlich, “Generation of Single-Stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA Locus,” Proc. Natl. Acad. Sci. (USA) 85: 7652-7656 (1988); Gyllensten, U. B. and Erlich, H. A. (1991) “Methods for generating single stranded DNA by the polymerase chain reaction” U.S. Pat. No. 5,066,584, Nov. 19, 1991. Asymmetric PCR differs from symmetric PCR in that one of the primers is added in limiting amount, typically 1/100 to ⅕ of the concentration of the other primer. Double-stranded amplicon accumulates during the early temperature cycles, as in symmetric PCR, but one primer is depleted, typically after 15-25 PCR cycles, depending on the number of starting templates. Linear amplification of one strand takes place during subsequent cycles utilizing the undepleted primer. Primers used in asymmetric PCR reactions reported in the literature, including the Gyllensten patent, are often the same primers known for use in symmetric PCR. Poddar (Poddar, S. (2000) “Symmetric vs. Asymmetric PCR and Molecular Beacon Probe in the Detection of a Target Gene of Adenovirus,” Mol. Cell Probes 14: 25-32 compared symmetric and asymmetric PCR for amplifying an adenovirus substrate by an end-point assay that included 40 thermal cycles. He reported that a primers ratio of 50:1 was optimal and that asymmetric PCR assays had better sensitivity that, however, dropped significantly for dilute substrate solutions that presumably contained lower numbers of target molecules.
Although asymmetric PCR has been known since 1988, it has not been extensively used as a technique because of the need to spend a great deal of time optimizing the experimental conditions for each amplicon. J. K. Ball and R. Curran (1997) “Production of Single-Stranded DNA Using a Uracil-N-glycosylase-Mediated Asymmetric Polymerase Chain Reaction Method,” Analytical Biochemistry 253: 264-267, states: “To ensure that asymmetric amplification occurs several replicate tubes containing different concentrations of each primer are set up, and for this reason the technique is not used extensively.”